Materials and Reagents

50 mL single-neck recovery flask (Ace Glass, catalog number: 945808)
Micropipettes, 10–100 µL (Eppendorf, catalog number: 13-684-251)
Pipette tips, 1–300 µL (Fisher Scientific, SureOne^TM micropoint pipette tips, catalog number: 02-707-410)
Serological pipettes, 5 mL (VWR, catalog number: 89130-886)
Disposable cuvettes (GMBH + Co KG, catalog number: 759075D)
Disposable folded capillary cell (Malvern, Zetasizer Nano Series, catalog number: DTS1070)
20% glucose solution (Gibco, catalog number: A2494001), stored at 4 °C
DEPC water (Invitrogen, catalog number: AM9916), stored at 4 °C
TurboFect^TM transfection reagent (Thermo Scientific^TM, catalog number: R0531), stored at 4 °C
Monogalactosyl-diacylglycerol (MGDG) (Avanti Polar Lipids, catalog number: 840523), stored at -20 °C
Digalactosyl-diacylglycerol (DGDG) (Avanti Polar Lipids, catalog number: 840524), stored at -20 °C
L-α-phosphatidic acid (PA) (Avanti Polar Lipids, catalog number: 840074), stored at -20 °C
IL-22 pMRNAXP vector (SBI, sequence XM_006513865.4), stored at -80 °C
mRNA synthesis kit (SBI, catalog number: MR-KIT-1), stored at -80 °C
200 proof ethanol (EtOH) (Decon Labs, catalog number: 2401); mix 350 mL of 100% EtOH with 150 mL autoclaved water for a solution of 70% EtOH
Dichloromethane (DCM) (Sigma, catalog number: 40042)

Equipment

Zetasizer Nano-ZS90 (Malvern Instruments Ltd., ZEN3690, serial number: MAL1181172)
Ultrasonicate cleaner (Branson Ultrasonics Corporation, Bransonic^®, model: 3510R-MTH)
Rotary evaporator (BUCHI, model: R-210)
Vacuum pump (BUCHI, model: V-700)

Software

Zetasize software 7.12 Copyright© 2002-2016 (Malvern Instruments Ltd.)

Procedure

Equipment setup
1. Set the ultrasonic bath temperature to 50 °C prior to the start of the experiment.
2. Set the rotatory evaporator water bath temperature to 50 °C prior to the start of the experiment.
3. Clean all glassware with 70% EtOH followed by 100% EtOH.
4. Sanitize the vacuum hood with UV light for 1 h.

Preparation of lipid solutions
1. Add 2.5 mL of 100% EtOH to 5.0 mg of MGDG to make a 2.0 mg/mL MGDG solution.
2. Add 2.5 mL of 100% EtOH to 5.0 mg of DGDG to make a 2.0 mg/mL DGDG solution.
3. Add 5.0 mL of 100% dichloromethane (DCM) to 25.0 mg of PA to make a 5.0 mg/mL PA solution.
4. Keep the above solutions stored at -20 °C until use.

Synthesis of IL-22/nLNPs
1. Mix 99 μL of MGDG, 66 μL of DGDG, and 66 μL of PA solutions in a 50 mL single-neck recovery flask.
2. Add 2.5 mL of 100 % EtOH to the lipid mixture.
3. Swirl the flask to mix the lipids and the EtOH mixture.
4. Attach the flask to the rotary evaporator and rotate for 3–5 min at a 50 torr pressure (Figure 1).
  
  Figure 1. Solvent evaporation by rotary evaporator
5. Release the vacuum when it reaches 50 torr pressure and transfer the flask to a UV-sanitized vacuum hood.
6. In a centrifuge tube, prepare a 5% glucose solution by mixing 1 mL of 20% glucose solution with 3 mL of RNase-free DEPC water.
7. Add 50 μL of in vitro transcription synthesized IL-22 mRNA (generated by using mRNA synthesis kit with custom designed IL-22 pMRNAXP vector as a template, following manufacturer’s instruction) and 50 μL TurboFect^TM transfection reagent to the 500 μL of 5% glucose solution and mix. The total volume will be 600 μL.
8. Incubate the mixture for 15 min at room temperature.
9. Transfer the solution to the single-neck recovery flask containing the lipid mixture.
10. Place the bottom of the recovery flask in the water contained in the ultrasonic bath and pipette up and down (approximately 100 times) until the mixture becomes cloudy (Figure 2).
11. Store the prepared nLNPs at 4 °C.
  
  Figure 2. Sonication of IL-22 mRNA and lipid mixture

Characterization of the nLNPs
Measure particle size
1. Place approximately 1.5 mL of nLNPs suspension in a cuvette and cover it with a plastic cap.
2. Place the cuvette in the cuvette holder of Malvern Zetasizer.
3. Set the software function to measure the size.
Measure zeta potential
1. Place approximately 1.0 mL of the nLNPs suspension in a capillary cell.
2. Place the cell in the cuvette holder of the Malvern Zetasizer.
3. Set the software function to measure the zeta potential.

Data analysis

Dynamic light scattering analysis showed that the average sizes for blank nLNPs (Figure 3) and IL-22/nLNPs (Figure 5) are 189.9 ± 69.3 and 184.2 ± 84.96 nm, respectively, based on triplicate measurement.
The zeta potential of blank nLNPs (Figure 4) and IL-22/nLNPs (Figure 6) are -5.51 ± 3.26 and -17.4 ± 7.63 mV, respectively, based on triplicate measurement.

Figure 3. Measurement of size in blank nLNPs. Particle size was determined to be 189.9 ± 69.3 nm in diameter.

Figure 4. Measurement of zeta potential in blank nLNPs. Surface zeta potential was measured as -5.51 ± 3.26 mV.

Figure 5. Measurement of size in IL-22/nLNPs. Particle size of IL-22/nLNPs was 184.2 ± 84.96 nm in diameter.

Figure 6. Measurement of zeta potential in IL-22/nLNPs. Surface zeta potential of IL-22/nLNPs was -17.4 ± 7.63.

Acknowledgments

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), grant numbers: RO1-DK-107739 (DM), RO1-DK-116306 (DM), and R01DK132891 (GA and SG). Department of Veterans Affairs (BX002526, DM). Dr. Merlin and Dr. Alpini are recipients of a Senior Research Career Scientist Award from the Department of Veterans Affairs.

Competing interests

The authors declare no conflicts of interest within the work.

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